In 1953, Watson and Crick suggested the concept of double stranded DNA. They had some significant discoveries. (1) DNA molecules were composed of two anti-parallel poly-nucleic acid chains. (2) There were rules for paring the four bases—chargaff et al. analyzed the base compositions of DNA molecules by chromatograph from many organisms, and found that the numbers of A and T were equal, while the numbers of C and G were also equal. So they suggest there exist four possible base pairs: A-T, T-A, G-C and C-G. (3) The connection of the two chains were through hydrogen bounds—the surface of the base pairs goes through and was roughly perpendicular to the axis. Two and three hydrogen bounds can form between the A-T and G-C pairs, respectively. Meanwhile, hydrophobic force also contributes to stabilize the DNA double helixes. (4) Because all the base pairs follow these rules, every chain can have random sequences. However, once the sequence of one of the chains is determined, the other one must have the corresponding nucleotide sequences.
As the DNA double helix is maintained by both hydrogen bound and hydrophobic force, factors, such as heat, pH, organic solvent, etc., which can destroy hydrogen and hydrophobic bounds, would denature DNA double helixes to random single chain threads. The annealing between denatured DNA single chains through pairing is called hybridization. Hybridization can occur between homologous DNA molecules as well as homologous DNA and RNA molecules. During hybridization, the two complementary single-stranded DNA chains form double-stranded hybrids through non-covalent bounds. When the sequence of one of the chains is known, the existence of its complementary chain in an unknown DNA sample can be detected through hybridization.
Based on the above principle, many gene products have been developed, among which gene sensor has many applications.
Recently, the research in the field of DNA sensor (DNA or nucleic acid sensor) has become the hot spot of research. Gene sensor, as a simple, fast, and cheap detection method, has many potential applications in the fields of molecular biology, medical analysis, and environment monitoring. It can also be applied to study DNA-drug and protein-protein interactions in addition to sequence analysis, mutation detection, gene detection, and clinical diagnostics.
The method for gene analysis to analyze DNA sequences in non-homogenous system is now through DNA hybridization. Hybridization can occur between homologous DNA molecules as well as homologous DNA and RNA molecules. During hybridization, the two complementary single-stranded DNA chains form double-stranded hybrids through non-covalent bounds. When the sequence of one of the chains is known, the existence of its complementary chain in an unknown DNA sample can be detected through hybridization. The most common method is to fix a single DNA chain with known sequence information on a solid surface, and use it to hybridize to the complementary single chains in the sample buffer to detect the existence of the wanted DNA molecule in the liquid phase.
Recently, such research is getting deeper and deeper, and there is great value in DNA detection through hybridization. The major applications rely in the fields of clinical diagnostics, forensic science, food industry, biochemistry, environment protection, etc. The application of using non-radioactive labels such as biotin, digosin, and fluorescent dyes has made the detection more convenient and safer. In particular, the application of PCR amplification has made the method very sensitive.
The traditional DNA hybridization reaction requires a labeling step to detect hybridization signals, which allow in situ detection, and can achieve high sensitivity. For example, PCR technology can reach the detection limit at the nmol/l range. Bioinformatics also provides means to detect a specific DNA sequence from a complex mixture of DNA. Because of using short wave fluorescence and co-focal microscopy, fluorescent labeling has become the routine method for detecting nano-amount of DNA molecules. Among the available fluorescent labeling methods, the device of DNA chip (gene chip or DNA microarray) only include the XYZ three-dimensional (3-D) parameters, such as the Affymetrix GeneChip. On these chips oligo nucleic probes are fix on glass surface, and their base composition and chain length can be represented by XYZ 3-D parameters. The single-stranded oligo target sequences are directly labeled with fluorescent dyes, hybridized to the probes on the chip to form double helixes, followed by the detection by using a scanner to obtain sample information. The U.S. Pat. No. 5,445,934 and U.S. Pat. No. 5,744,305, disclosed the technologies and device of using photolithography to fabricate high-density DNA probes on a DNA chip. In such device the hybridization between all the fixed probes on the chips and the other DNA molecules in the sample buffer was carried out at the same temperature. Because of the differences between the probe lengths, base composition (GC content), the Tm (melting temperature), which represents the temperature when 50% of the probes and their targets are separated, are different. Therefore, the optimum hybridization temperatures are different for each probe. Because of such discrepancy in hybridization temperature, the accuracy of results cannot be guaranteed, and single base mismatch cannot be detected. To overcome such disadvantages, U.S. Pat. No. 6,238,868 described a type of microarrays by introducing electric filed as a free parameter to expedite the hybridization procedure. However, this technology is complicated, high cost, and requires the labeling of oligo target sequences or hybridizing with another reporter probes with fluorescent dyes. As a result it cannot guarantee the accuracy of results, and limited the application of hybridization microarrays.
Recently, some researchers begin to use non-labeling methods to analyze gene sequences. The most popular one is the DNA biosensor system. These biosensors can be categorized into three classes: (1) optical biosensor, which can be further divided into three classes of fluorescent optical fiber gene sensor, surface enhance Raman gene probe and surface plasmon resonance gene sensor, (2) electro-chemistry biosensor, and (3) piezo gene biosensor. Further information of their specificity and sensitivity is still waiting.